EP4293103A1 - Bifidobacterium lactis und anwendung davon - Google Patents

Bifidobacterium lactis und anwendung davon Download PDF

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Publication number
EP4293103A1
EP4293103A1 EP22752363.6A EP22752363A EP4293103A1 EP 4293103 A1 EP4293103 A1 EP 4293103A1 EP 22752363 A EP22752363 A EP 22752363A EP 4293103 A1 EP4293103 A1 EP 4293103A1
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EP
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Prior art keywords
lactis
bifidobacterium animalis
animalis subsp
pharmaceutical composition
food composition
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EP22752363.6A
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English (en)
French (fr)
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EP4293103A4 (de
Inventor
Yunfeng DUAN
Ye Zhang
Hua LIANG
Zhi Liu
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Wisbiom Beijing Biotechnology Co Ltd
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Wisbiom Beijing Biotechnology Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/51—Bifidobacterium
    • A23V2400/515—Animalis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure relates to the technical field of microorganisms, in particular to a Bifidobacterium animalis subsp. lactis and use thereof.
  • intestinal bacteria can affect the physical development of the host, and some bacteria can mediate the physical development of the host by affecting growth hormone (GH)/insulin-like growth factor-1 (IGF-1). Both germ-free mice and antibiotic-treated mice exhibited reduced growth, which was associated with reduced levels of growth hormone releasing peptide, growth hormone and IGF-1. In addition, in both mice and humans, microbial deficiency leads to maturation and growth restrictions, such as chronic malnutrition, anorexia nervosa, growth retardation, short stature, and even developmental abnormalities of the nervous and immune systems.
  • GH growth hormone
  • IGF-1 insulin-like growth factor-1
  • Insulin-like growth factor 1 (IGF-1), as a hormone with a known effect on bone growth, significantly increases in serum levels as well as in liver and adipose tissue with increasing intestinal microbial colonization. However, after antibiotic treatment, intestinal microbiota is greatly reduced, which can reduce serum IGF-1 and inhibit bone formation. Supplementation with the microbial metabolite short-chain fatty acids (SCFA) after antibiotic treatment restored IGF-1 and bone mass to normal levels. In addition, germ-free mice had lower IGF-1 levels. However, treatment with beneficial microbes modestly increased bone mineral density, increased IGF-1 levels and prevented bone loss. Insulin-like growth factor 1 (IGF-1) is a growth factor that affects endocrine and paracrine/autocrine pathways of bone growth.
  • SCFA short-chain fatty acids
  • Exogenous IGF-1 can promote the longitudinal growth of the femur.
  • IGF-1 was observed to affect the maturation of the growth plate and the formation of secondary ossification centers in the absence of chondro-specific insulin-like growth factor I receptor (Igf1r).
  • Igf1r insulin-like growth factor I receptor
  • Liver-specific IGF-1-deficient mice have been found to have a 75% reduction in serum IGF-1 but still exhibit relatively normal physical development, suggesting that local IGF-1 can also promote bone growth.
  • IGF-1 can promote osteoblasts (including affecting bone formation and bone resorption, respectively). Therefore, the alteration of intestinal microbiota can promote bone formation and resorption, leading to net bone growth, and the microbiota can promote bone growth and remodeling by inducing IGF-1.
  • Short-chain fatty acids (SCFA) produced by direct supplementation of probiotics, prebiotics, or microbial fermentation of cellulose may induce an increase in IGF-1, which in turn affects bone growth and health.
  • intestinal microbiota can regulate brain function and behavior through the gut-brain axis.
  • the intestinal microbiota and the brain communicate bi-directionally through the autonomic nervous system, enteric nervous system, immune system, olfactory system, enteroendocrine signals, neurotransmitters, branched chain amino acids, bile acids, short-chain fatty acids, spinal cord, hypothalamic-pituitary-adrenal axis, peptidoglycan and other pathways and mediators.
  • the bidirectional communication between the intestinal microbiota and the central nervous system, that is microbiota-gut-brain axis, can affect the neurodevelopment and function of animals, thereby affecting social behavior.
  • This bidirectional effect of the microbiota-gut-brain axis is affected by internal factors such as gender and genetics, as well as external factors such as environment, diet, genetics, and stress. Therefore, the intestinal microbiota is involved in the pathogenesis of a variety of nervous system related diseases. At the same time, psychological and behavioral responses can in turn affect the composition or function of the intestinal microbiota. It has been proved that interventions targeting the intestinal microbiota are expected to treat diseases such as anxiety, depression, schizophrenia, ADHD, transient tic disorder, Parkinson's disease, Alzheimer's disease, and social disorders such as autism.
  • ADHD attention-deficit/hyperactivity disorder
  • age-mismatched attention deficit reduced attention span
  • excessive activity regardless of occasions, emotional impulsivity, and often accompanied by cognitive impairment, conduct disorder, and learning difficulties, etc.
  • the ADHD has a high incidence, which has a significant negative impact on the academic, family and social life of patients.
  • the present disclosure provides a Bifidobacterium animalis subsp. lactis and use thereof.
  • the Bifidobacterium animalis subsp. lactis can improve intestinal metabolic disorder and normalize the composition of intestinal microbiome so as to effectively suppress obesity, promote the infant and youth physical development, and promote their mental development.
  • the Bifidobacterium animalis subsp. lactis can also effectively improve intestinal permeability and reduce levels of LPS and D-lactic acid in the blood, so as to play a role in the prevention and treatment of a mental disorder.
  • Such mental disorder comprises anxiety, depression, attention-deficit/hyperactivity disorder, autism, autistic disorder, schizophrenia, hepatic encephalopathy, anorexia, Tourette syndrome, and Asperger syndrome.
  • a Bifidobacterium animalis subsp. lactis which is Bifidobacterium animalis subsp. lactis BL-11, wherein the Bifidobacterium animalis subsp. lactis BL-11 has an accession number of CGMCC No. 20847.
  • a Bifidobacterium animalis subsp. lactis preparation wherein the preparation is a solid bacterial powder or a liquid beverage, and the Bifidobacterium animalis subsp. lactis is Bifidobacterium animalis subsp. lactis described in the first aspect of the present disclosure.
  • a seventh aspect of the present disclosure there is provided use of the above Bifidobacterium animalis subsp. lactis in the preparation of a food composition or a pharmaceutical composition for promoting infant and youth mental development.
  • the Bifidobacterium animalis subsp. lactis BL-11 has a content of 1-25 parts by weight, preferably 1-15 parts by weight, relative to 100 parts by weight of the food composition or the pharmaceutical composition; the Bifidobacterium animalis subsp. lactis BL-11 is used in a form of live bacteria of the Bifidobacterium animalis subsp. lactis BL-11, inactivated bacteria of the Bifidobacterium animalis subsp. lactis BL-11 or extracts from the Bifidobacterium animalis subsp. lactis BL-11.
  • the Bifidobacterium animalis subsp. lactis BL-11 has a viable count of 1.0 ⁇ 10 6 -1.5 ⁇ 10 12 CFU/g, preferably 3.0 ⁇ 10 10 -5.0 ⁇ 10 11 CFU/g.
  • the food composition is one or more of fermented milk, cheese, milk containing beverage, solid beverage, and milk powder.
  • the Bifidobacterium animalis subsp. lactis BL-11 is administered at a dose of 2.0 ⁇ 10 6 -1.5 ⁇ 10 11 CFU/kg/day, preferably 3.0 ⁇ 10 4 -8.0 ⁇ 10 10 CFU/kg/day, based on a weight of a human body.
  • the mental disorder comprises anxiety, depression, attention-deficit/hyperactivity disorder, autism, autistic disorder, schizophrenia, hepatic encephalopathy, anorexia, Tourette syndrome, and Asperger syndrome.
  • the food composition or the pharmaceutical composition further comprises one or more of skimed milk powder, trehalose, fructooligosaccharide, lactose, glucose, sucrose, L-sodium ascorbate, L-malic acid, and L-lactic acid.
  • the food composition or the pharmaceutical composition further comprises a flavoring agent, a sweetener, a thickener, a stabilizer, a surfactant, a lubricant, an acid neutralizing agent, a dispersant, a buffer solution or a buffer, a debitterizing agent, pH stabilizer, a preservative, a desugaring agent and/or a coloring agent, such as, lactitol, sorbitol, maltitol, aspartame, stevia rebaudiana, siraitia grosvenorii, sucralose, xylitol, vanilla, chocolate, fruit flavors, artificial flavors, or a mixture or combination thereof.
  • a flavoring agent such as, lactitol, sorbitol, maltitol, aspartame, stevia rebaudiana, siraitia grosvenorii, sucralose, xylitol, vanilla, chocolate, fruit flavors, artificial flavors, or a mixture or combination thereof.
  • the food composition or the pharmaceutical composition further comprises a vitamin, a mineral and/or a dietary supplement or a prebiotic nutrient, or at least one prebiotic, wherein, optionally, the prebiotic comprises inulin, artichoke extract, chicory root extract, jerusalem artichoke root extract, fructooligosaccharide, galactooligosaccharide, isomalto-oligosaccharide, xylooligosaccharide, stachyose, mannose oligosaccharide, arabinose oligosaccharide, resistant dextrin, resistant starch, or a mixture or combination thereof.
  • the prebiotic comprises inulin, artichoke extract, chicory root extract, jerusalem artichoke root extract, fructooligosaccharide, galactooligosaccharide, isomalto-oligosaccharide, xylooligosaccharide, stachyose, mannose oligo
  • the food composition or the pharmaceutical composition further comprises ubiquinone (CoQ10), lycopene, ⁇ -carotene, tryptophan, vitamin B6, vitamin B12, or a mixture or combination thereof.
  • ubiquinone CoQ10
  • lycopene lycopene
  • ⁇ -carotene tryptophan
  • vitamin B6, vitamin B12 or a mixture or combination thereof.
  • the food composition or pharmaceutical composition further comprises probiotics, wherein, optionally, the probiotics comprise microorganisms or bacteria or bacterial components that are cultured or extracted from faeces, and optionally, the bacteria or bacterial components comprise or are derived from Lactobacillus, Bifidobacterium, Escherichia coli (E. coli), Prevotella, Faecalibacterium, Blautia, Bacteroidetes, Firmicutes, and an equivalent, or a mixture or combination thereof.
  • probiotics comprise microorganisms or bacteria or bacterial components that are cultured or extracted from faeces
  • the bacteria or bacterial components comprise or are derived from Lactobacillus, Bifidobacterium, Escherichia coli (E. coli), Prevotella, Faecalibacterium, Blautia, Bacteroidetes, Firmicutes, and an equivalent, or a mixture or combination thereof.
  • the Bifidobacterium animalis subsp. lactis BL-11 has a content of 0.5-20 parts by weight, preferably 1-15 parts by weight, relative to 100 parts by weight of the food composition or the pharmaceutical composition; the Bifidobacterium animalis subsp. lactis BL-11 is used in a form of live bacteria of the Bifidobacterium animalis subsp. lactis BL-11, inactivated bacteria of the Bifidobacterium animalis subsp. lactis BL-11 or extracts from the Bifidobacterium animalis subsp. lactis BL-11; and the Bifidobacterium animalis subsp. lactis BL-11 has a viable count of 1.0 ⁇ 10 6 -1.5 ⁇ 10 12 CFU/g, preferably 3.0 ⁇ 10 10 -5.0 ⁇ 10 11 CFU/g.
  • the food composition is one or more of fermented milk, cheese, milk containing beverage, solid beverage, and milk powder.
  • the Bifidobacterium animalis subsp. lactis BL-11 is administered at a dose of 2.0 ⁇ 10 9 -1.5 ⁇ 10 11 CFU/kg/day, preferably 3.0 ⁇ 10 4 -8.0 ⁇ 10 10 CFU/kg/day, based on a weight of a human body.
  • the food composition or the pharmaceutical composition may be a powder, a tablet, a liquid, a gum, a soft candy, a tablet candy, a yogurt, a milk, a cheese, an ice cream, a frozen food, a health food, a drug, or a feed.
  • the Bifidobacterium animalis subsp. lactis BL-11 of the present disclosure can improve intestinal permeability and reduce levels of LPS and D-lactic acid in the blood.
  • the Bifidobacterium animalis subsp. lactis BL-11 of the present disclosure can be used to promote autonomous active activity, improve the memory function of disposable avoidance response, and improve anhedonia caused by stress stimuli.
  • probiotics provided in the present disclosure can effectively improve intestinal metabolic disorder and normalize the composition of intestinal microbiome, promote the infant and youth physical development, and can improve intestinal permeability and reduce levels of LPS and D-lactic acid in the blood, so as to play a role in the prevention and treatment of a mental disorder.
  • Such mental disorder comprises anxiety, depression, attention-deficit/hyperactivity disorder, autism, autistic disorder, schizophrenia, hepatic encephalopathy, anorexia, Tourette syndrome, and Asperger syndrome.
  • Bifidobacterium animalis subsp. lactis BL-11 classified as Bifidobacterium animalis subsp. lactis, was deposited at China General Microbiological Culture Collection Center (CGMCC, Address: NO.1 Beichen West Road, Chaoyang District, Beijing 100101, China; Institute of Microbiology, Chinese Academy of Sciences) on October 10, 2020, and it was assigned accession number CGMCC No. 20847.
  • CGMCC General Microbiological Culture Collection Center
  • a Bifidobacterium animalis subsp. lactis which is Bifidobacterium animalis subsp. lactis BL-11, wherein the Bifidobacterium animalis subsp. lactis BL-11 has an accession number of CGMCC No. 20847.
  • a Bifidobacterium animalis subsp. lactis preparation wherein the preparation is a solid bacterial powder or a liquid beverage, and the Bifidobacterium animalis subsp. lactis is Bifidobacterium animalis subsp. lactis described in the first aspect of the present disclosure.
  • a seventh aspect of the present disclosure there is provided use of the above Bifidobacterium animalis subsp. lactis in the preparation of a food composition or a pharmaceutical composition for promoting infant and youth mental development.
  • the Bifidobacterium animalis subsp. lactis BL-11 has a content of 1-25 parts by weight, preferably 1-15 parts by weight, relative to 100 parts by weight of the food composition or the pharmaceutical composition; the Bifidobacterium animalis subsp. lactis BL-11 is used in a form of live bacteria of the Bifidobacterium animalis subsp. lactis BL-11, inactivated bacteria of the Bifidobacterium animalis subsp. lactis BL-11 or extracts from the Bifidobacterium animalis subsp. lactis BL-11.
  • the Bifidobacterium animalis subsp. lactis BL-11 has a viable count of 1.0 ⁇ 10 6 -1.5 ⁇ 10 12 CFU/g, preferably 3.0 ⁇ 10 10 -5.0 ⁇ 10 11 CFU/g.
  • the food composition is one or more of fermented milk, cheese, milk containing beverage, solid beverage, and milk powder.
  • the Bifidobacterium animalis subsp. lactis BL-11 is administered at a dose of 2.0 ⁇ 10 6 -1.5 ⁇ 10 11 CFU/kg/day, preferably 3.0 ⁇ 10 4 -8.0 ⁇ 10 10 CFU/kg/day, based on a weight of a human body.
  • the mental disorder comprises anxiety, depression, attention-deficit/hyperactivity disorder, autism, autistic disorder, schizophrenia, hepatic encephalopathy, anorexia, Tourette syndrome, and Asperger syndrome.
  • the food composition or the pharmaceutical composition further comprises one or more of skimed milk powder, trehalose, fructooligosaccharide, lactose, glucose, sucrose, L-sodium ascorbate, L-malic acid, and L-lactic acid.
  • the food composition or the pharmaceutical composition further comprises a flavoring agent, a sweetener, a thickener, a stabilizer, a surfactant, a lubricant, an acid neutralizing agent, a dispersant, a buffer solution or a buffer, a debitterizing agent, pH stabilizer, a preservative, a desugaring agent and/or a coloring agent, such as, lactitol, sorbitol, maltitol, aspartame, stevia rebaudiana, siraitia grosvenorii, sucralose, xylitol, vanilla, chocolate, fruit flavors, artificial flavors, or a mixture or combination thereof.
  • a flavoring agent such as, lactitol, sorbitol, maltitol, aspartame, stevia rebaudiana, siraitia grosvenorii, sucralose, xylitol, vanilla, chocolate, fruit flavors, artificial flavors, or a mixture or combination thereof.
  • the food composition or the pharmaceutical composition further comprises a vitamin, a mineral and/or a dietary supplement or a prebiotic nutrient, or at least one prebiotic, wherein, optionally, the prebiotic comprises inulin, artichoke extract, chicory root extract, jerusalem artichoke root extract, fructooligosaccharide, galactooligosaccharide, isomalto-oligosaccharide, xylooligosaccharide, stachyose, mannose oligosaccharide, arabinose oligosaccharide, resistant dextrin, resistant starch, or a mixture or combination thereof.
  • the prebiotic comprises inulin, artichoke extract, chicory root extract, jerusalem artichoke root extract, fructooligosaccharide, galactooligosaccharide, isomalto-oligosaccharide, xylooligosaccharide, stachyose, mannose oligo
  • the food composition or the pharmaceutical composition further comprises ubiquinone (CoQ10), lycopene, ⁇ -carotene, tryptophan, vitamin B6, vitamin B12, or a mixture or combination thereof.
  • ubiquinone CoQ10
  • lycopene lycopene
  • ⁇ -carotene tryptophan
  • vitamin B6, vitamin B12 or a mixture or combination thereof.
  • the food composition or pharmaceutical composition further comprises probiotics, wherein, optionally, the probiotics comprise microorganisms or bacteria or bacterial components that are cultured or extracted from faeces, and optionally, the bacteria or bacterial components comprise or are derived from Lactobacillus, Bifidobacterium, Escherichia coli (E. coli), Prevotella, Faecalibacterium, Blautia, Bacteroidetes, Firmicutes, and an equivalent, or a mixture or combination thereof.
  • probiotics comprise microorganisms or bacteria or bacterial components that are cultured or extracted from faeces
  • the bacteria or bacterial components comprise or are derived from Lactobacillus, Bifidobacterium, Escherichia coli (E. coli), Prevotella, Faecalibacterium, Blautia, Bacteroidetes, Firmicutes, and an equivalent, or a mixture or combination thereof.
  • the Bifidobacterium animalis subsp. lactis BL-11 has a content of 0.5-20 parts by weight, preferably 1-15 parts by weight, relative to 100 parts by weight of the food composition or the pharmaceutical composition; the Bifidobacterium animalis subsp. lactis BL-11 is used in a form of live bacteria of the Bifidobacterium animalis subsp. lactis BL-11, inactivated bacteria of the Bifidobacterium animalis subsp. lactis BL-11 or extracts from the Bifidobacterium animalis subsp. lactis BL-11; and the Bifidobacterium animalis subsp. lactis BL-11 has a viable count of 1.0 ⁇ 10 6 -1.5 ⁇ 10 12 CFU/g, preferably 3.0 ⁇ 10 10 -5.0 ⁇ 10 11 CFU/g.
  • the food composition is one or more of fermented milk, cheese, milk containing beverage, solid beverage, and milk powder.
  • the Bifidobacterium animalis subsp. lactis BL-11 is administered at a dose of 2.0 ⁇ 10 9 -1.5 ⁇ 10 11 CFU/kg/day, preferably 3.0 ⁇ 10 4 -8.0 ⁇ 10 10 CFU/kg/day, based on a weight of a human body.
  • the food composition or the pharmaceutical composition may be a powder, a tablet, a liquid, a gum, a soft candy, a tablet candy, a yogurt, a milk, a cheese, an ice cream, a frozen food, a health food, a drug, or a feed.
  • the Bifidobacterium animalis subsp. lactis BL-11 of the present disclosure can improve intestinal permeability and reduce levels of LPS and D-lactic acid in the blood.
  • the Bifidobacterium animalis subsp. lactis BL-11 of the present disclosure can be used to promote autonomous active activity, improve the memory function of disposable avoidance response, and improve anhedonia caused by stress stimuli.
  • This example is used to illustrate Bifidobacterium animalis subsp. lactis BL-11 and its performance characteristics.
  • Bifidobacterium is a genus of bacteria that are not usually acid-fast.
  • the tolerance of the artificial gastric fluid and intestinal fluid of Bifidobacterium animalis subsp. lactis BL-11 of the present invention was tested, and at the same time, the currently stored Bifidobacterium animalis subsp. lactis Bb-XX which has excellent acid resistance and can survive in the gastrointestinal tract was used as a control.
  • the survival rate of strain BL-11 in artificial gastric fluid was shown in Table 3.
  • the viable survival rate of strain Bb-XX in artificial gastric fluid was 44.7% at 1 hour of treatment and 29.5% at 3 hours of treatment.
  • Bifidobacterium animalis subsp. lactis BL-11 of the present invention had a viable survival rate of 86.2% at 1 hour of treatment and 39.5% at 3 hours of treatment.
  • the survival test results of the strain BL-11 in artificial intestinal fluid was shown in Table 3.
  • the survival rate of Bifidobacterium animalis subsp. lactis BL-11 in artificial intestinal fluid was 67.5% at 1 hour of treatment, and the survival rates of the two strains were 49.4% and 32.1% after 3 hours of treatment, respectively.
  • the Bifidobacterium animalis subsp. lactis BL-11 of the present invention was inoculated into MRS Liquid medium and cultured anaerobically at 37 °C for 48 hours.
  • the viable count of the Bifidobacterium animalis subsp. lactis BL-11 in the culture medium was 3.7 ⁇ 10 9 CFU/mL.
  • the culture stock solution was orally administered to the mice at a dose of 20.0 mL/kg body weight by gavage for 3 consecutive days, after which the mice were observed for an additional 7 days.
  • mice Healthy male BALB/C mice, aged 6-8 weeks old, and weighted 16-18g, were fed and watered adlibitum at room temperature (25 ⁇ 2°C), relative humidity (55 ⁇ 2) %, and 12h/12h light/dark.
  • the results showed that compared with the control group, the group treated with the culture stock solution of Bifidobacterium animalis subsp. lactis BL-11 shows no toxic reaction or death, and there was no significant difference in the weight gain of mice between the two groups (p > 0.05).
  • the antibiotic sensitivity of Bifidobacterium animalis subsp. lactis BL-11 was evaluated by SN/T 1944-2007 "Determination of bacterial resistance in animals and their products". The evaluation results showed that, the Bifidobacterium animalis subsp. lactis BL-11 was positive for Ampicillin, penicillin G, Erythromycin, Chloramphenicol, Clindamycin, Vancomycin and Tetracycline, etc, which met the requirements of the European Food Safety Authority (EFSA) standard for the evaluation of resistance of edible bacteria.
  • EFSA European Food Safety Authority
  • the Bifidobacterium animalis subsp. lactis BL-11 does not contain exogenous antibiotic resistance genes and is safe to eat.
  • This example is used to illustrate the functional characteristics of Bifidobacterium animalis subsp. lactis in promoting physical development.
  • Microbiota promotes bone formation as well as resorption, resulting in net bone growth.
  • the microbiota induces hormone-like insulin growth factor 1 (IGF-1), which promotes bone growth and remodeling.
  • IGF-1 hormone-like insulin growth factor 1
  • SCFA Short-chain fatty acids
  • mice Female BALB/c mice, aged two month old, were treated with antibiotics and probiotics and maintained under SPF conditions. 30 mice were combined and randomly assigned to treatment groups to minimize cage effects.
  • mice were treated with the drinking water mixed with antibiotic mixture for 2 weeks to consume microorganisms, and then the mice were divided into three groups with 10 mice in each group.
  • One group was fed normally as a control group (CK group), and the other two groups were fed with water mixed with probiotics BL-11 or Bb-XX as probiotic groups (including BL-11 or Bb-XX group).
  • the mice in the three groups were fed for four weeks.
  • 3% (g/100mL) sucrose was added to water in all groups to ensure palatability, as specified in the animal facility.
  • the aqueous solution was freshly prepared and changed twice a week. After four weeks, mice were sacrificed, and serum was then prepared from blood samples collected by cardiac puncture using a serum separator tube, and the murine IGF-1 standard ABTS ELISA development kit (PeproTech) was used.
  • mice and antibiotic-treated mice exhibited reduced growth, reduced levels of growth hormone releasing peptide, growth hormone and IGF-1, and disordered intestinal microbes, leading to growth restriction in mice.
  • the serum IGF-1 levels of the mice treated with antibiotics and then treated with probiotics were measured. Results are shown in Fig. 5 , and show that the serum IGF-1 levels of the mice treated with antibiotics and then treated with probiotics were increased.
  • BL-11 can significantly increase the serum IGF-1 levels of the mice ( P ⁇ 0.01), than, and the effect of BL-11 was higher than that of Bb-XX.
  • Diversity of intestinal microbiota a diversity index analysis showed that there was no significant difference in diversity index between BL-11 and Bb-XX groups.
  • BL-11 group has a slightly higher Shannon index than Bb-XX group, but failed to achieve significant difference ( P >0.05), and the results are shown in Fig. 6 .
  • the analysis of the two beneficial bacteria in the intestine of mice in the two groups showed that the use of BL-11 could significantly increase a ratio of Bifidobacteria to Lactobacillus in the intestine, and the results are shown in Fig. 7 .
  • Heat map analysis shows that at the genus level, Faecalibacterium, Lachnospira, Lachnospiraceae_UCG_004, Sutterella were increased in the BL-11 group.
  • the increase of these bacteria may be related to the increase of IGF-11 in serum.
  • This example is used to illustrate the preparation of BL-11 bacterial powder and its use in the production of food.
  • Bifidobacterium animalis subsp. lactis BL-11 provided in the present invention was cultured anaerobically in MRS Broth liquid medium. After primary culture, secondary culture and expanded culture, the fermentation broth was incubated at 37°C for 24 hours, centrifuged at 4°C and 3000 rpm for 10 minutes, the bacteria were collected, washed with phosphate buffer solution (PBS), freeze-dried with skimed milk, and stored below -20°C.
  • PBS phosphate buffer solution
  • the BL-11 bacterial powder prepared in this example may be used for foods, drugs, health products or animal feeds.
  • the food can be fermented milk, cheese, milk containing beverage, solid beverage, milk powder and other common food or health food.
  • the suggested dose of Bifidobacterium animalis subsp. lactis BL-11 for human use may be 1.0 ⁇ 10 3 -1.0 ⁇ 10 10 CFU/kg body weight/day, more preferably 1.0 ⁇ 10 4 -1.0 ⁇ 10 9 CFU/kg body weight/day.
  • This example is used to illustrate functional characteristics of promoting physical development in human clinical research.
  • Subjects and recruitment 65 patients with Prader-Willi syndrome aged 11 months to 16 years were openly recruited and randomly assigned to two groups treated with probiotics or placebo to perform a 12-week randomized, double-blind, placebo-controlled experiment.
  • Inclusion criteria patients with genetically confirmed PWS, without taking any form of probiotics within four weeks, taking a stable drug for at least four weeks; with no planned pharmacologic or psychological interventions during the experiment, willing to provide fecal samples in a timely manner, willing to participate in the study and interview process and without other genetic diseases, pregnancy or breastfeeding conditions.
  • Written informed consent was obtained from the parents or legal guardians of the subjects, as required by the IRB, for the study protocol, which was conducted in accordance with the Declaration of Helsinki.
  • Randomized and blinded testing A randomized, double-blind, placebo controlled design was used. Subjects was randomly assigned and concealed them by a statistician who was not part of the team, so that each unidentified subject generated a random sample number. The coded probiotic and placebo with the same appearance were provided by BEIJING HUAYUAN BIOTECHNOLOGY RES INSTITUTE, to ensure that allocation was concealed and blind spots were maintained. These patients were randomly assigned to receive daily strip bags of the probiotic Bifidobacterium animalis subsp. lactis BL-11 (6 ⁇ 10 10 CFU) or placebo. Body weight, height, ASQ-3, ABC, SRS-2 and CGI-1 were compared between the two groups at 6 and 12 weeks of treatment. The CGI comprises two single-item measurements that assess: (a) psychopathology severity from 1 to 7 (CGI-S) and (b) change in symptoms before and after treatment assessed on a seven-point scale from start to end (CGI-I).
  • the probiotic group is treated with powder Bifidobacterium animalis subsp. lactis BL-11 in the form of strip bag.
  • Each bag of Bifidobacterium animalis subsp. lactis supplement contained 3 ⁇ 10 10 colony-forming units (CFU).
  • Placebo was maltodextrin in the same bag as the strip bag of Bifidobacterium animalis subsp. lactis, similar in color, taste, and flavor to the strip bag of Bifidobacterium animalis subsp. lactis.
  • maltodextrin had minimal side effects
  • maltodextrin also had minimal adverse effects.
  • Fecal samples were collected with DNA/RNA shielded fecal collection tubes (Zymo, Cat#R1 10 1) containing 1mL of preservation solution, transported to the laboratory by ice pack, and then frozen at -80 °C.
  • DNA was extracted using the TIANmap fecal DNA kit (TIANGEN, catalog number DP328) according to the manufacturer's instructions, and DNA samples were carefully quantified using a Nanodrop spectrophotometer. The A260/A280 ratio was measured to confirm the yield of highly purified DNA.
  • the DNA samples were frozen at -20 °C for use.
  • 16S rRNA V3-V4 libraries were constructed by two rounds of PCR using the following primers:
  • the PCR reaction (95 °C for 2min, followed by 25 cycles of 95 °C for 30s, 55 °C for 30s, and 72 °C for 30s) was performed, followed by a final extension at 72 °C for 5min.
  • the PCR product was purified with 1 ⁇ KAPA AMPure (KAPA, catalog number KK8002). Then, the product was subjected to a second PCR reaction step (95 °C for 2min, followed by eight cycles of 95 °C for 30s, 55 °C for 30s and 72 °C for 30s, and finally extension at 72 °C for 5min).
  • PCR products were purified by 1 ⁇ KAPA AMPure and quantified by real-time PCR using the Bioanalyzer DNA kit.
  • CGI Clinical Global Impression
  • Gastrointestinal symptoms were assessed on the basis of the total number of GI symptoms including constipation, diarrhea, abdominal pain, flatulence, hematochezia, nausea, dysphagia, loss of appetite, dyspepsia, and acid regurgitation, at baseline.
  • the present disclosure uses false discovery rate (FDR) to adjust the multiple comparison results. Secondary outcomes were analyzed using methods similar to those used for the primary outcomes. In addition, linear regression was performed to examine the correlation between clinical index and microbiome composition.
  • FDR false discovery rate
  • Sequencing reads were filtered with QIIME2 (v2019.10) for quality control. Default parameters were denoised using Deblur, and abundance tables of samples were obtained by amplicon sequence variants (ASVs). Alpha diversity was calculated using QIIME2. Bray-Curtis distance was used to characterize ⁇ diversity of the microbiome. ASVs were assigned using a Sklearn-based classifier classification, wherein the classifier was trained on sequences with 99% similarity to Greengenes v13.8. It was confirmed by Kruskal-Wallis test that there were significant differences between the placebo and probiotic groups in the relative abundance of microbial phylum, genus, and alpha diversity. False discovery rate (FDR) based on Benjamini-Hochberg (BH) adjustment was used for multiple comparisons.
  • FDR False discovery rate
  • BH Benjamini-Hochberg
  • PICSRUSt2 was used to infer the functional content of microorganisms based on the abundance table of ASVs, and then to generate the Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologs (KO), enzyme taxonomic numbers and pathway abundance tables. Differences in rates between the probiotic and placebo groups were analyzed using a permutation based nonparametric test, and the most significant difference features were plotted with Calour. All raw data from 16s rRNA Illumina amplicon sequencing have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA, PRJNA643297).
  • NCBI National Center for Biotechnology Information
  • Table 4 provides an overview of demographic characteristics and gastrointestinal (GI) symptoms of comorbidities in 65 participants, with no between-group differences observed ( P >0.05). 47.5% of subjects in the study population showed one or more GI symptoms. The percentage of patients with GI symptoms was 37.4% lower in the probiotics group than in the placebo group, but the difference was not significant ( P >0.05). Table 4 Bifidobacterium animalis subsp.
  • probiotics group had an average increase of 2.58cm, which was significantly higher than the placebo group, P ⁇ 0.05
  • the probiotics group had a greater weight loss over time than the placebo group, but the between-group difference was not significant ( Fig. 10 ).
  • Linear mixed-effects model analysis showed the probiotics group had a trend toward greater improvement than the placebo group in psychometric scores (including ASQ-3, ABC, SRS, and RRB), but the differences were not significant ( P >0.05).
  • the intestinal microbiome composition of PWS individuals was differentially enriched in two groups during the intervention.
  • the overall relative abundance levels of specific bacteria are shown in Fig. 12 .
  • Fig. 14 In order to characterize changes in the abundance of bacteria that may be clinically significant during the intervention, the present disclosure presented fold changes for genera and families of several selected bacteria, as shown in Fig. 14 .
  • the probiotics group at weeks 6 and 12, the relative abundance of Lachnospiraceae ND3007, Ruminococcaceae UCG-003, Streptococcus mutans, Comamonadaceae, Alistipes and Rothia was reduced compared with baseline.
  • beneficial bacterial genera such as Bifidobacterium, Lactobacillus, and Prevotella 9 increased significantly at 12 weeks compared with baseline ( Fig. 15 ).
  • lactis BL-11 may have a role in improving the infant and youth mental development, behavior, or emotional state.
  • This example is used to illustrate the preparation of BL-11 lyophilized bacterial powder.
  • the storage solution of BL-11 bacteria was resuscited in a water bath at 37°C until all the liquid in the frozen storage tube melted.
  • the MRS Medium was streaked in sections, and the bacteria were cultured anaerobically at 37°C for 12-24 hours. Single colonies were selected and inoculated into anaerobic culture tubes containing liquid medium, sealed, and incubated at 37°C for 6-12 hours.
  • the OD600 value of bacterial solution stopped growing, the fermentation was stopped.
  • the bacterial solution was fermented and incubated at a constant temperature of 40°C for 6-12 hours with stirring.
  • the constant pH of the bacterial solution was maintained at 5.4 ⁇ 0.5.
  • the OD600 value of the bacterial solution stopped increasing, the fermentation was stopped immediately.
  • This example is used to illustrate BL-11 lyophilized bacterial powder for use in the production of food.
  • the lyophilized bacterial powder produced from Bifidobacterium animalis subsp. lactis BL-11 provided in the present invention can be used in ordinary food or health food such as yogurt, fermented milk, cheese.
  • the dose of Bifidobacterium animalis subsp. lactis BL-11 for preparing yogurt is 1.0 ⁇ 10 6 -1.0 ⁇ 10 8 CFU/kg, more preferably 1.2 ⁇ 10 7 -1.5 ⁇ 10 8 CFU/kg.
  • the prepartion method is a direct addition method or a post addition method.
  • the lyophilized bacterial powder is used as fermentation strain, and added in proportion after raw milk was sterilized and reduced to the appropriate temperature, and fermentation was then carried out at 40-43 °C for 10-48 hours to obtain a product of fermentation. After stirring and mixing, the product of fermentation is divided into cups or bags as a finished product.
  • the lyophilized bacterial powder is added in a certain proportion after the fermentation of yoghurt, and then stirring, mixing and divided into cups or bags as a finished product.
  • This example is used to illustrate BL-11 lyophilized bacterial powder for use in the production of dietary nutritional supplements, and probiotics.
  • the lyophilized bacterial powder produced from Bifidobacterium animalis subsp. lactis BL-11 provided by the present invention can be used for the production of probiotics.
  • Probiotics lyophilized powder comprises 0.5-30 parts of BL-11 lyophilized bacterial powder, 5-20 parts of bacteria such as Lactobacillus fermentium, Lactobacillus helveticus, Lactobacillus reusei, Lactobacillus plantarum, bifidobacterium adolescentis, Bifidobacterium breve, and Bifidobacterium longum, 20-70 parts of prebiotics such as galactooligosaccharide, fructooligosaccharide, and inulin, 5-10 parts of nutrients such as GABA, tryptophan, lycopene, ⁇ -carotene, vitamin B6, vitamin B12, coenzyme Q10, taurine, pectin, ⁇ -glucan, fucose, carrageenan
  • the probiotics is added at a total viable count of 2.0 ⁇ 10 6 -2.0 ⁇ 10 10 CFU/g, preferably 3 ⁇ 10 7 -3 ⁇ 10 10 CFU/g.
  • Each of the other bacteria is added at a dose of 1 ⁇ 10 6 -3 ⁇ 10 9 CFU/g.
  • the preparation process includes: weighting raw materials in proportion, mixing, packing and quality inspection, etc.
  • This example is used to illustrate the effects of BL-11 lyophilized bacterial powder on improving intestinal permeability and behavior.
  • Gut microbiota and gut-brain axis play a two-way communication effect in the regulation of stress response.
  • Microbiota communicates with the gut-brain axis through different mechanisms. It interacts directly with mucosal cells, influences brain development and behavior through immune cells, and through contacting with nerve endings. Stress in the brain can also have an effect via GBA on the gut microbiome, which is responsible for GI dysfunction and dysregulation.
  • Stress responses also affect the synthesis of microbial by-products and precursors that enter the brain through the blood and hindbrain regions, release cytokines through mucosal immune cells, release gut hormones such as serotonin (5-HT) through enteroendocrine cells, or through afferent nervous pathway, including the enteric nervous system.
  • cytokines through mucosal immune cells
  • gut hormones such as serotonin (5-HT)
  • enteroendocrine cells or through afferent nervous pathway, including the enteric nervous system.
  • the probiotic preparation comprises lyophilized powder containing BL-11, ⁇ -carotene, vitamin B6, vitamin B12, coenzyme Q10 and maltodextrin, and the control group was maltodextrin.
  • the experimental mice were treated with 10 billion CFU/mouse/day by gavage.
  • mice Twenty C57BL/6J mice aged 6 weeks old were housed in four cages with five mice per cage, and fed and watered adlibitum. Food and water intake were recorded twice weekly. The mice were divided into control group and probiotics group, with 10 mice in each group. After one week of acclimatization, the mice were randomly divided into two groups: model group and probiotics intervention group. The growth conditions of the experimental mice were: ambient temperature (23 ⁇ 2)°C, relative humidity (50 ⁇ 10) %, and light mode (12h dark /12h light).
  • the model of chronic stress caused by physical stimulation was adopted.
  • the specific stimulation methods included: (1) fasting and water deprivation for 24 hours; (2) continuous light for 24 hours; (3) clamping tail with an iron clip from 1cm of the tail tip for 3min/ time; (4) restraint stress with circular sleeve restraint for 2h/ day.
  • One or two different stress stimulation were used every day, and the stimulation time was not fixed for 4 weeks.
  • control group had a decreased preference for the aqueous solution of sucrose (49.63% ⁇ 15.79), while after supplementation of probiotics, the preference for the aqueous solution of sucrose was increased (68.79% ⁇ 12.34), indicating that probiotics could improve the anhedonia caused by stress stimulation.
  • Step-down instrument test box (DTT-2 mouse step-down, Institute of Materia Medica, Chinese Academy of Medical Sciences) was used.
  • the test box is about 120cm long, 12cm wide and 30cm high, and is made of plexiglass. There were six chambers, each 12cm long, 12cm wide, and 30cm high.
  • the test box allowed six mice to be tested at same time.
  • the bottom of the test box is covered with a copper gate, which is connected to the power supply through a wire, and the current voltage is set to 36V.
  • An insulation table (a pentagon-shaped wooden block with a long diameter of 5.7cm, a short diameter of 4.5cm and a high of 4.8cm) was placed on the copper gate in the test box as a safety area for animals to avoid electric shocks.
  • the test box was connected to a computer automatic recording system.
  • the mice were put into the step-down instrument test box to acclimate for 5 minutes, and then gently placed on the table.
  • the copper gate was electrified. When the mice jumped off the table and touched the copper gate, they would receive electric shocks. The normal avoidance response was to jump on the table and return to the safety zone to escape the electric shock.
  • This learning was carried out for 5 minutes, and the number of electric shocks (the number of errors) within 5 minutes was recorded, which was regarded as the learning score.
  • memory tests were performed. The mice were placed on the table, and the time from staying on the table to receiving the first electric shock as the latency period, and the number of errors within 5 minutes (the number of electric shocks that all limbs of the mice touched the copper gate at the same time) were recorded as the evaluation indexes of memory function.
  • the probiotics group Compared with the normal control group, the probiotics group has a significantly shortened latency period in the step-down test ( P ⁇ 0.05). There was no significant difference in the number of errors in step-down test between the two groups, but the probiotics group showed a decreasing trend in the number of errors. The results showed that the probiotics could improve the memory function of one-time avoidance response in mice (see Table 5).
  • Table 5 Effect of probiotics on memory function of avoidance response Number of errors (times) Latency period (S) Control group 1.45 ⁇ 0.82 268.32 ⁇ 50.63 Probiotics group 1.07 ⁇ 0.87 248.05 ⁇ 82.33 P value 0.0348 0.0782
  • the open field test analysis system is used to observe and study the neuropsychological changes of animals, and various behaviors of animal. For example, animals are afraid of the new open environment, after entering the open environment, in which animals mainly act in the peripheral area and less in the central area. However, the exploratory characteristics of animals promote their motivation to act in the central area, and the resulting anxiety can also be observed. It was used to assess the level of voluntary active activity and anxiety in animals.
  • mice were moved to the open field test room 60min before the experiment to acclimate to the environment in advance.
  • the mice were removed from the cage and placed in the open field test device in the behavioral experimental station (the box has length ⁇ width ⁇ height of 100cm ⁇ 100cm ⁇ 40cm, blue inner and bottom sides, and the camera placed directly above the central area).
  • the mice were placed in a fixed position in the central area, with the head fixed to one side each time, and the light mask curtain was quickly pulled.
  • the recording system was turned on, the nine-square mode was selected with ratio of central area of 0.5, and the camera above the open field device and the connected monitor were opened.
  • the measurements included movement time, total distance, percentage of time spent in the central area (time spent in the central area LPMM s), percentage of horizontal movement in the central area (distance of horizontal movement in the central area and L distance of horizontal movement), percentage of horizontal movement in the four sides area (distance of horizontal movement in the four sides area and L distance of horizontal movement) and percentage of horizontal movement in the four corners area (distance of horizontal movement in the four corners area and L distance of horizontal movement).
  • the times of standing and grooming were recorded, and then the environment in the box was cleaned with 75% alcohol. After the alcohol volatilized and became odorless, the next mouse was measured. The results are shown in Table 6.
  • mice Total movement distance (cm) Time spent in the central area (S) Number of entry into the central area Number of standing Number of grooming Control group 383.34 ⁇ 189.82 6.45 ⁇ 2.14 3.22 ⁇ 2.56 9.32 ⁇ 6.78 4.88 ⁇ 1.68 Probiotic group 486.74 ⁇ 212.71 9.87 ⁇ 2.77 5.84 ⁇ 2.97 13.87 ⁇ 5.94 6.29 ⁇ 2.13
  • D-lactic acid is a metabolite of bacterial fermentation, and can be produced by a variety of intestinal bacteria. Even if D-lactate is ingested from food, it is rarely absorbed into the blood under normal conditions. Mammals do not possess an enzymatic system for the rapid degradation of D-lactate. Therefore, when intestinal mucosal permeability is increased, a large amount of D-lactic acid produced by bacteria in the gut passes through the damaged mucosa into the blood, increasing the level of D-lactic acid in the blood.
  • Lactic acid has D-form and L-form.
  • the normal human body only has L-lactate, and bacteria and other microorganisms can produce D-lactic acid.
  • the level of D-lactic acid in blood can timely reflect the degree of intestinal mucosal damage and changes in permeability. It can be used for the auxiliary evaluation of intestinal infection, endotoxemia, systemic inflammatory reaction, recurrent fever, vomiting, etc.
  • LPS Lipopolysaccharide
  • bacterial endotoxin is a component of the cell wall of Gram-negative bacteria.
  • LPS is a toxic substance for animals.
  • the structure of LPS is consisted of three parts: glycolipid domain-lipid A, sugar residue short chain-core oligosaccharide, and hypervariable polysaccharide domain-O antigen.
  • the structure of LPS determines its agonist/antagonist effect on TLR4.
  • LPS binds to TLR4/MD-2 receptor complex and activates different signaling pathways through myd88-dependent or TRIF-dependent pathways.
  • the expression of TLR in different parts of intestinal epithelial cells is different, preventing the inflammatory response caused by LPS and fighting against pathogenic bacteria.
  • Bacterial translocation is the process by which live intestinal bacteria enter the body from the gut through the epithelial mucosa. Bacteria can enter the lymphatic system through the mesenteric lymph nodes and circulate systemically. They can also enter the circulation, causing bacteremia, and can locate in tissues. Bacterial translocation can result in small intestinal bacterial overgrowth, intestinal injury, and shock. Any stress response including psychological and physiological tress response that leads to intestinal permeability, could potentially lead to bacterial translocation.
  • LPS is associated with the pathogenesis of many diseases, such as IBD and enterocolitis and other intestinal diseases, and even Parkinson's and Alzheimer's diseases. LPS can not only enter the blood, but also enter and remain in the brain for a lifetime, which may cause Alzheimer's disease.
  • the level of LPS in the blood can reflect the permeability of the intestine, and the normal intestinal barrier does not allow LPS to enter.
  • the higher level of LPS in the blood indicates the translocation of intestinal bacteria or LPS into the blood, indicating the increase of intestinal permeability and the increase of intestinal leakage symptoms.
  • the amount of LPS in the blood can also indicate the inflammatory response and stress state. Excessive LPS can cause abnormal immune system, chronic or acute inflammatory response, and acute inflammation such as fever and pain. It can be used for the auxiliary evaluation of intestinal infection, endotoxemia, systemic inflammatory reaction, recurrent fever, vomiting, mental illness, stress response, etc.
  • This example is used to illustrate the effect of BL-11 lyophilized bacterial powder on the intestinal microbial composition.
  • Amplification of bacterial 16S rRNA gene Diluted genomic DNA was used as a template, and specific primers with Barcode were used depending on the region selected for sequencing. Bacterial universal primers 341F (CCTAYGGGRBGCASCAG, SEQ ID NO.3) and 806R (GGACTACNNGGGTATCTAAT, SEQ ID NO.4) were used to amplify the V3-V4 region of the bacterial 16S rRNA gene. 100ng of extracted DNA was subjected to PCR at 56°C for strand renaturation, PCR was performed as follows: denaturation at 94°C for 4 min, followed by 30 cycles of 94°C for 30s, 56°C for 30s, and 72°C for 1min.
  • the library was constructed by Illumina TruSeq DNA PCR-Free Library Preparation Kit. The constructed library was subjected to Qubit quantification and library detection. After the library was qualified, microbiota sequencing was performed using the Illumina HiSeq2500 PE250 sequencing platform.
  • This example is used to illustrate the results of an intervention with a probiotic consisting of BL-11 bacteria in 3 years and 6 months old children diagnosed with autism spectrum disorder (ASD).
  • the probiotic was administered in the form of oral lyophilized powder at a dose of 50 billion CFU twice daily for a 90-day cycle.
  • the children's defecation habit changed from once every 3-5 days to once every 1-2 days.
  • Parents reported that the children's vocabulary, socialization and eye contact frequency increased, and the number of produced words increased by 3-5 words.
  • the children have increased frequency of active feedback of their feelings, and number of unsolicited requests.
  • the repetitive actions were reduced relative to the no-intervention condition. The parents chose to let the children continue to take probiotic, and still observe and record the continuous improvement.
  • This example is used to illustrate the improvement of a probiotic consisting of BL-11 bacteria on attention deficit hyperactivity disorder (ADHD).
  • ADHD attention deficit hyperactivity disorder
  • the patients were from Hebei Children's Hospital. The doctor diagnosed ADHD according to the Diagnostic and Statistical Manual of Mental Disorders. The patients were 6.5 years old, were hyperactive and impulsive, and also had chronic constipation and abdominal discomfort, excluded from schizophrenia, affective disorder, epilepsy, and other organic diseases.
  • the patient was treated with oral probiotics lyophilized powder at a dose of 60 billion CFU twice a day.
  • the powder was taken with warm water after breakfast and dinner.
  • the treatment lasted for 12 weeks.
  • the Conners Parent Questionnaire was used to assess indexes, including behavior, physical, anxiety, learning, hyperactivity, etc. The results showed that impulsivity, anxiety, and hyperactivity scores decreased, defecation function improved from once every 3-4 days to once or twice every day, sometimes once every 2-3 days, defecation volume increased, and bloating and pain gradually disappeared. The children's overall mental condition improved significantly, and parents were satisfied with the results.
  • Table 8 Results of the Conners Parent Questionnaire before and after treatment Behavior Learning Psychosomatic Impulse Anxiety Hyperactivity Before treatment 1.6 2.1 0.35 1.34 0.52 1.38 After treatment 1.2 1.8 0.29 1.32 0.35 1.23

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